Translocation of proteins to phospholipid membranes involves specific protein structures and can be triggered by the presence in the interface of a non-phospholipid lipid "second messenger," like diacylglycerol The long-range goal of this research is to understand how interactions between phospholipids and non-phospholipid second messengers regulate peripheral protein binding to interfaces and the subsequent expression of catalytic activity. Its focus is pancreatic triacylglycerol lipase (PTL) and its cofactor protein, colipase (COL), for which lipids like diacylglycerols are activators as well as lipase substrates. PTL and two other members of its gene family, lipoprotein lipase and hepatic lipase, are the primary regulators of the distribution of lipids to and from peripheral tissues. Hence, they are highly relevant targets in the treatment of diseases of lipid homeostasis like obesity and atherosclerosis. To function properly, the lipid-binding motif of the N-terminal domain of PTL must bind to the lipid-water interface in its catalytically-efficient or 'open' conformation. The role of the C-terminal domain of PTL in this is unclear. In the fluid lipid interfaces at which PTL functions, phospholipids and lipase substrates form dynamic complexes that mix with uncomplexed lipids. We hypothesize that complexes inhibit the rate of protein adsorption and, hence, lipolysis. We further hypothesize that once PTL and COL bind, they rearrange lipid species in the interface to help overcome the inhibition. Apolipoprotein C-II, the cofactor for lipoprotein lipase, appears to act similarly to COL despite its lack of structural homology. To test these hypotheses, we propose 1) to define the role of lipid complexes in regulating the association of PTL's domains and COL to interfaces. To accomplish this we will use radiometric and fluorometric methods to measure initial rates of COL and PTL domain binding as a function of interfacial composition and packing; 2) to determine the ability of each of the three lipid associating motifs of PTL and COL to perturb lipid lateral organization. To accomplish this we will fluorimetrically determine the extent to which each bound protein motif is able to laterally redistribute lipid species in interfaces; 3) to determine the interfacial requirements for the binding of PTL's catalytic domain in the catalytically-efficient conformation. To accomplish this we will chemically and spectroscopically determine how PTL catalytic domain binding and conformation are regulated; 4) to define the functional similarities of the PTL-COL lipolytic system with the serum lipoprotein lipase-apolipoprotein C-II system. To accomplish this the techniques used in aims 1-3 will be applied to these proteins. With its unique focus on the role of lipid interfacial structure in regulating protein binding, this research will provide a specific mechanistic understanding of how cofactor proteins enable lipolysis to occur at physiologically relevant interfaces. More broadly, it will help to explain how lipid second messengers regulate protein translocation associated with signaling events in cells.